CN113388673B - 长非编码rna carmen的应用 - Google Patents
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Abstract
本发明提供了一种长非编码RNA CARMEN的应用,具体为检测长非编码RNA CARMEN表达的试剂在制备用于预测或辅助诊断先天性心脏病的产品中的应用。本发明涉及生物医学技术领域,本发明发现长非编码RNA CARMEN在患有先天性心脏病的新生儿脐血中表达显著下调且体外实验发现在胚胎干细胞向心肌细胞分化过程中敲低CARMEN的表达后,心肌细胞的数量也降低,表明长非编码RNA CARMEN可用于预测或辅助诊断先天性心脏病。
Description
技术领域
本发明涉及生物医学技术领域,尤其涉及长非编码RNA CARMEN的应用,具体涉及一种检测长非编码RNA CARMEN表达的试剂在制备用于预测或辅助诊断先天性心脏病的产品中的应用。
背景技术
长链非编码RNA,也称为长非编码RNA(Long non-coding RNA,lncRNA),是一类长度大于200nt,的非编码RNA分子。lncRNA一般无蛋白编码能力。lncRNA的保守性较低,lncRNA的表达具有组织特异性以及时空特异性。lncRNA占总RNA的比例可达4%-9%。
长非编码RNA的作用范围广泛,作用机制非常复杂。长非编码RNA本身不编码蛋白,但可以调控基因的表达,例如参与表观遗传调控以及转录和转录后调控等。长非编码RNA在发育、机体内平衡和维持细胞命运中发挥重要作用。近些年来,长非编码RNA(Longnoncoding RNAs,lncRNA)的研究热度不断上升,其功能也越来越多地被研究和报道。例如,Mayuresh Anant Sarangdhar等报道亲本遗传的长链非编码RNA Cyrano参与斑马鱼神经发育,美国德州大学MD安德森癌症中心的研究人员发现上调lncRNA LINC00538(YIYA)可促进乳腺癌中的糖酵解,细胞增殖和肿瘤生长,Mandal和他团队发现存在于白细胞中的长非编码RNA分子HOTAIR具有在细菌存在下向这些细胞发出信号以激活免疫应答的能力。
先天性心脏病是胎儿期心脏及大血管发育异常所致的先天性畸形,是小儿最常见的心脏病。目前关于先天性心脏病的诊断方法主要有影像学诊断(例如胸部X线)、心电图、超声心动图等。寻找先天性心脏病的相关基因以及研究先天性心脏病调控异常的转录因子或者其他分子已成为当前的心血管分子生物学领域的研究焦点。关于lncRNAs在先天性心脏病中的研究目前很少有相关报道,鉴于对lncRNAs的研究将有助于分析lncRNAs的表达调控机制,并有望为复杂疾病的预测、诊断,甚至治疗提供新的依据,因此,探索潜在的lncRNAs在先天性心脏病中的功能和作用机制是十分有益的。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供长非编码RNA CARMEN的应用,具体为一种检测长非编码RNA CARMEN表达的试剂在制备用于预测或辅助诊断先天性心脏病的产品中的应用。
为了实现上述目的,本发明提供的技术方案如下:
检测长非编码RNA CARMEN表达的试剂在制备用于预测或辅助诊断先天性心脏病的产品中的应用,所述长非编码RNA CARMEN的核苷酸序列如以下SEQ ID No.1所示:
agctcatataaggtaaaaggcagagtggcctctgtggccagggagccgcaggcaagggactaaggggaggggggctcagtgccagctgcttaaaaatgcccctgtggcagcgaggggcaccagaggctgggtctaattagttgagaagcagtgacacccccaaccactccccaaacaggctggctcccgtctccaggccccaaggagccacacctggaccagaccccaggaaagacatgtggtggaaaccctacggccctggagcccaaggagagcaacggctgtaacaagtgcacaggcaagggcgctagctggtgggagccaccccgccatgctgatgtcagagaagcaagaactctggagaagcagcctcctgggaccagaggagggccagcagcaggcagcccggagacagaactctcacccacgcctctccaagcctcccaacagaagacagaggtcccccacagccagagacatttcctgaagacatggggaacacagaggcagaaacagcccatccacccaggagctgtcccccacactgccgggagccggcacccagagccgccaggtaaaactgaggccacctggttcaacatcacctttcacagaaggggaagcagccacagaaagaagggcctcgttaagaagtggaacctgggacccccaagcggtgtctctcatcctgactggggatccagagtaggagggagcctttggtggggttggagtcccgccacaggccaccagagcggagcagcgcagcgccctgtctcccagcctgaggtgcagtgctgcatctctggtcagttgggagtctgagatgaagcactgtagctcaggaagagagaagttgttctgcagccatcagcctggaagtgcctggctggtgggccttcttgcagtagcttcccctggagaagaggaaaagcaaaccttcattgagacccaagcggtctctcctgtgctctgtgacaataataaagttccagcccttggcaaaaaaaaaa。
长非编码RNA CARMEN是一种超强增强子相关的lncRNA。
在一个实施方案中,所述产品包括试剂盒、芯片或试纸。
在一个实施方案中,所述试剂为对所述长非编码RNA CARMEN的表达量进行定量的试剂。
在一个具体的实施方案中,所述RNA CARMEN的表达量在先天性心脏病患儿的血液中表达显著下调;优选地,所述RNA CARMEN的表达量在先天性心脏病的新生儿脐血中表达显著下调。
在一个实施方案中,所述试剂为实时荧光定量PCR检测试剂。
在一个实施方案中,所述试剂包含检测所述长非编码RNA CARMEN表达量时使用的特异性引物组。
本发明提供了所述特异性引物组在检测所述长非编码RNA CARMEN表达量的试剂或试剂盒中的应用。
在一个实施方案中,所述特异性引物组的上下游引物序列如SEQ ID No.2和SEQID No.3所示;具体地,所述上游引物为:5’-CAGTGCTGCATCTCTGGTCA-3’(SEQ ID No.2);所述下游引物为5’-CCAGGGGAAGCTACTGCAAG-3’(SEQ ID No.3)。
在一个实施方案中,所述试剂还包含检测内参基因的上下游引物。
在一个实施方案中,所述内参基因为GAPDH。
在一个实施方案中,检测所述内参基因的上下游引物如SEQ ID No.4和SEQ IDNo.5所示。具体地,所述内参基因的上游引物为5’-TTCTTTTGCGTCGCCAGCC-3’;所述内参基因的下游引物为5’-GACGGTGCCATGGAATTTGCC-3’。
在一个实施方案中,所述试剂盒还包括RNA提取缓冲液、总RNA反转录反应液和qPCR反应液。在一个实施方案中,所述试剂盒还包括SYBR Green荧光染料。
本发明的有益效果:
本发明从新生儿脐血样本中抽提RNA后逆转录,实时荧光定量法检测了样本中CARMEN的表达,结果显示CARMEN在患有先天性心脏病的新生儿脐血中表达下调。此外,本发明的体外实验也发现胚胎干细胞向心肌细胞分化过程中敲低CARMEN的表达后,心肌细胞的数量也降低,提示CARMEN在心肌生成中有重要作用。
本发明还提供了用于先天性心脏病辅助诊断的试剂和试剂盒,具有良好的临床应用前景。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的实时荧光定量PCR技术检测lncRNA CARMEN在脐血中表达情况;Control:健康对照组;CHD:先天性心脏病患儿组;*P<0.05 vscontrol;
图2为本发明实施例提供的胚胎干细胞向心肌细胞分化过程中CARMEN的表达变化;D0、2、4、6、8、10:分化的不同时间点。*P<0.05,**P<0.01 vsD0 group;
图3为本发明实施例提供的实时荧光定量PCR技术检测GapmeR对CARMEN的敲低作用;A:Control,正常分化组;B:GapmeR,CARMEN敲低组;**P<0.01 vsD0,n=3;
图4为本发明实施例提供的免疫荧光检测心肌细胞的标志蛋白cTnI的表达水平;A:Control,对照组;B:GapmeR,CARMEN敲低组;红色:cTnI;蓝色:DAPI。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1.实时荧光定量PCR法检测CARMEN在先天性心脏病新生儿的脐血中的表达
材料与方法:
收集20例先天性心脏病新生儿及20例正常对照新生儿的脐血,所有受试者均已经签署临床研究知情同意书。
纳入标准:出生后经心脏彩照诊断为先天性心脏病的患儿。
排除标准:(1)伴有心外系统畸形患儿;
(2)伴有心脏系统之外的其它疾病。
抽提RNA,经逆转录成cDNA后上机进行实时荧光定量PCR检测。
1.RNA的提取
使用RNA提取专用试剂盒(RNeasy kit,QIAGEN)提取RNA。先将培养孔板的细胞培养基去除,PBS清洗两次,每孔再加入450μL RLT缓冲液(1mL RLT缓冲液中配比10μLβ-巯基乙醇),吹打充分裂解后收集到淡紫色的QIAshredder spin column中,14,000rpm离心2min,取上清至2mL的无酶EP管,加0.5倍体积无水乙醇,吹打充分混匀,再将样品(约650μL)转移至RNeasy spin column,再14,000rpm离心1min,弃滤出物,再加入700μL RW1缓冲液至柱中,再14,000rpm离心1min,清洗滤膜,弃滤出物和收集管,将柱转移至新收集管,加入500μL RPE缓冲液中,再14,000rpm离心1min,清洗柱子滤膜,弃滤出物;清除残留的RPE缓冲液,最后将RNeasy spin column转移至1.5mL收集管,在滤膜上在加入30μL无RNA酶水,14,000rpm再次离心1min,洗脱RNA后即可收集RNA样品。
2.逆转录cDNA
逆转录体系1:Total RNA1μg,10mM dNTP mix 1μL,50μM oligo(dT)201μL,及适量RNase Free dH2O补足总体积为10μL。
反应条件:将反应体系1在65℃孵育5min后放置冰上1-2min。每管加入10μL反应体系2,吹打混合并低速离心后50℃孵育50min,85℃,5min后将样品放置于冰上,在每个样品中加入1μL RNase H,37℃孵育20min,-20℃保存。
3.实时荧光定量PCR检测
引物序列设计:
CARMEN的正向引物为5’-CAGTGCTGCATCTCTGGTCA-3’(SEQ ID No.2),反向引物为5’-CCAGGGGAAGCTACTGCAAG-3’(SEQ ID No.3)。
用于内参照的GAPDH的正向引物为5’-TTCTTTTGCGTCGCCAGCC-3’(SEQ ID No.4),反向引物为5’-GACGGTGCCATGGAATTTGCC-3’(SEQ ID No.5)。
PCR反应体系(共20μL):
正向引物(上游引物)(10μM) | 0.4μL |
反义引物(下游引物)(10μM) | 0.4μL |
模板cDNA | 2μL |
SYBR Green Mix | 10μL |
无核酶水 | 7.2μL |
PCR反应条件:
Cycle 1:95℃,2min;
Cycle 2:(40cycles):95℃,30s→58℃-65℃(设置梯度温度),30s→72℃,30s;
Cycle 3:95℃,1min;
Cycle 4:55℃,1min;
Cycle 5:(80cycles):Melting curve(55-95℃);
Cycle 6:4℃,hold。
采用相对定量分析法(2-ΔΔCt)分析,以细胞内管家基因GAPDH的PCR的Ct值作为相对标准,计算目的基因的相对表达量。
结果分析:
荧光定量PCR的结果显示,LncRNACARMEN在疾病组的表达量较正常对照组下调,且差异具有统计学意义(P<0.05,图1),说明CARMEN的表达水平可以反映先天性心脏病的状态。
实施例2.胚胎干细胞向心肌细胞分化过程中CARMEN的表达水平
采用胚胎干细胞系H9作为研究对象,分析H9在向心脏谱系细胞分化过程中,CARMEN的表达变化。
材料与方法:
体外无饲养层方法培养H9(WA09)人胚胎干细胞(ESC)。将H9在Matrigel包被的组织培养板上无饲养层培养,基础培养基为多潜能干细胞培养基,常规诱导向心肌分化。
具体步骤为:采用EDTA消化H9细胞,将细胞悬浮液均匀分装铺板至已包被Matrigel的六孔板中。D0日加入心肌细胞分化培养基A溶液(RPMI1640基础培养基+B27+6μMChir99021)培养48小时后,PBS洗涤一次,D2日加入心肌细胞分化培养基B溶液(RPMI1640基础培养基+B27+5μM IWP2)继续分化48小时,D4日PBS洗涤后加入心肌细胞分化培养基C溶液(RPMI1640基础培养基+B27+10μM SB431542),并隔日换液培养,分别在D0、2、4、6、8、10日收集细胞,提取总RNA,按实施例1方法逆转录cDNA,并进行荧光实时定量PCR检测。
结果分析:
荧光实时定量PCR显示LncRNACARMEN在D2日开始表达上升,D4日达到高峰,然后开始缓慢下降,D8至D10维持在一个稳定的高水平。说明在胚胎干细胞向心肌细胞分化过程中,CARMEN的表达水平是增加的(P<0.01,图2)。
实施例3.敲低CARMEN后的心肌细胞的分化情况
采用丹麦Exiqon公司LNATMlongRNA GapmeRs敲低CARMEN的表达水平,然后检测CARMEN表达下降后,心肌细胞的分化情况。
材料与方法:
在六孔板中铺上H9细胞,使得细胞数量为2.4*105/孔,加入3μL的CARMNE特异性的GapmeR(终浓度50nM),孵育24h后更换为新鲜心肌分化培养基,分化至D10日采用免疫荧光检测心肌细胞阳性标志物cTnI的表达情况。荧光实时定量PCR检测CARMEN的表达水平。荧光实时定量PCR方法同前。
免疫荧光检测:
用PBS洗3次细胞,加入4%多聚甲醛固定20min,再用PBS洗3次,5min/次。加入0.1%Triton X-100通透化处理20min。加入4%BSA封闭液中封闭1h,同样PBS洗3次,接着与anti-cTnI一抗在4℃孵育过夜。次日在PBS中洗涤后3次,10分钟/次,用羊抗鼠二抗孵育玻片1h,注意避光,用含DAPI的抗体进行核染色,封片剂进行封片处理,以上每次抗体孵育后均用PBS洗涤3次,10min/次,随后用荧光显微镜拍照。
结果分析:
与正常分化的对照组相比,CARMEN敲低组的CARMEN水平是降低的(图3),同时cTnI阳性细胞数量也明显减少(图4),说明心肌细胞的分化受CARMEN水平的调控,低水平的CARMEN会导致胚胎干细胞向心肌细胞分化能力降低。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 广东省人民医院;广东省心血管病研究所
<120> 长非编码RNA CARMEN的应用
<130> PA21008056
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1013
<212> DNA
<213> lncRNA CARMEN
<400> 1
agctcatata aggtaaaagg cagagtggcc tctgtggcca gggagccgca ggcaagggac 60
taaggggagg ggggctcagt gccagctgct taaaaatgcc cctgtggcag cgaggggcac 120
cagaggctgg gtctaattag ttgagaagca gtgacacccc caaccactcc ccaaacaggc 180
tggctcccgt ctccaggccc caaggagcca cacctggacc agaccccagg aaagacatgt 240
ggtggaaacc ctacggccct ggagcccaag gagagcaacg gctgtaacaa gtgcacaggc 300
aagggcgcta gctggtggga gccaccccgc catgctgatg tcagagaagc aagaactctg 360
gagaagcagc ctcctgggac cagaggaggg ccagcagcag gcagcccgga gacagaactc 420
tcacccacgc ctctccaagc ctcccaacag aagacagagg tcccccacag ccagagacat 480
ttcctgaaga catggggaac acagaggcag aaacagccca tccacccagg agctgtcccc 540
cacactgccg ggagccggca cccagagccg ccaggtaaaa ctgaggccac ctggttcaac 600
atcacctttc acagaagggg aagcagccac agaaagaagg gcctcgttaa gaagtggaac 660
ctgggacccc caagcggtgt ctctcatcct gactggggat ccagagtagg agggagcctt 720
tggtggggtt ggagtcccgc cacaggccac cagagcggag cagcgcagcg ccctgtctcc 780
cagcctgagg tgcagtgctg catctctggt cagttgggag tctgagatga agcactgtag 840
ctcaggaaga gagaagttgt tctgcagcca tcagcctgga agtgcctggc tggtgggcct 900
tcttgcagta gcttcccctg gagaagagga aaagcaaacc ttcattgaga cccaagcggt 960
ctctcctgtg ctctgtgaca ataataaagt tccagccctt ggcaaaaaaa aaa 1013
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
cagtgctgca tctctggtca 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
ccaggggaag ctactgcaag 20
<210> 4
<211> 19
<212> DNA
<213> 人工序列
<400> 4
ttcttttgcg tcgccagcc 19
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<400> 5
gacggtgcca tggaatttgc c 21
Claims (9)
1.定量检测长非编码RNA CARMEN表达量的试剂在制备用于预测或辅助诊断先天性心脏病的产品中的应用,所述长非编码RNA CARMEN的核苷酸序列如SEQ ID No. 1所示。
2.根据权利要求1所述的应用,其特征在于,所述产品包括试剂盒、芯片或试纸。
3.根据权利要求1所述的应用,其特征在于,所述试剂为实时定量PCR检测试剂。
4.根据权利要求3所述的应用,其特征为:所述试剂包含检测所述长非编码RNA CARMEN表达量时使用的特异性引物组。
5.根据权利要求4所述的应用,其特征在于,所述特异性引物组的上下游引物序列如SEQ ID No. 2和SEQ ID No. 3所示。
6.根据权利要求4或权利要求5所述的应用,其特征在于,所述试剂还包含检测内参基因的上下游引物。
7.根据权利要求6所述的应用,其特征在于,所述内参基因为GAPDH。
8.根据权利要求7所述的应用,其特征在于,检测所述内参基因的上下游引物如SEQ IDNo. 4和SEQ ID No. 5所示。
9.根据权利要求2所述的应用,其特征在于,所述试剂盒还包括RNA提取缓冲液、总RNA反转录反应液和qPCR反应液。
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